Large 3d porous scaffolds made of active hydroxyapatite obtained by biomorphic transformation of natural structures and process for obtaining them

ABSTRACT

The present disclosure relates to a hydroxyapatite obtained from porous wood, having high compressive strength and dimensions suitable for clinical applications. The porous wood has a porosity of between about 60% and about 95%, said porosity being measured after subjecting the wood to a step of pyrolysis, and is selected from among rattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnut wood. The hydroxyapatite may be substituted with one or more ions such as magnesium, strontium, silicon, titanium, carbonate, potassium, sodium, silver, gallium, copper, iron, zinc, manganese, europium, gadolinium. Also disclosed is a bone substitute comprising hydroxyapatite obtained from porous wood. The bone substitute is utilized for the substitution and regeneration of a bone or a bone portion, preferably for bones subjected to mechanical loads, such as long bones of the leg and arm, preferably the tibia, fibula, femur, humerus and radius. The invention relates also to a process for manufacturing a biomorphic hydroxyapatite scaffold from wood.

The present disclosure relates to hydroxyapatite obtained from porouswood. In particular, the present disclosure relates to a biomorphichydroxyapatite scaffold obtained from porous wood for use as a bonesubstitute. The disclosure relates also to a process to convert woodinto a biomorphic hydroxyapatite scaffold which can be used as bonesubstitute.

BACKGROUND

Current ceramic processing and engineering are based on awell-established sequence of processes enabling the production of large3D bodies. More specifically, innovative ceramic phases can besynthesized as powders, where specific features such stoichiometry/ionsubstitutions, nanosize, and surface activity, are responsible forspecific functionalities. The ceramic processing currently used toobtain macroscopic 3D ceramic bodies with adequate shape and porosityimplies thermal treatment (sintering) of the synthesized ceramic powderssuitably formed into a 3-D body (to consolidate the body). All thesesteps are needed to obtain 3D ceramics with adequate physicochemical andmechanical properties, most of which are degraded during theabove-mentioned ceramic process (particularly the sintering treatment).The serious limitations in the development of functional ceramicmaterial, associated with the current ceramic process, impede furtherprogress in the field.

Nowadays, with the evolution of modern society, technological productsare assuming a steadily increasing role in the life and productivity ofpeople, so that there is a strong need for smart tools able to providesolutions to complex and personalized demands, in various fields ofapplication, e.g. health, environment, energy. Therefore, there is awide consensus that new approaches are needed for the repeatable andmassive production of macroscopic devices with complex structuralorganization at the macro-scale but, at the same time with a complexstructure defined at the nanoscale, and even at the crystal scale. Suchmacro and nano-structures are relevant to induce non-trivial, but smartfunctional effects.

With respect to the above-mentioned issues regarding ceramic materials,a paradigmatic change is required in order to develop large highlyactive ceramics with complex micro and macro-structures.

Bone scaffolds, with particular focus to the regeneration of large,load-bearing bone defects, can be taken as a representative examplesince they should be porous 3-D ceramics with high bioactivity, in orderto be able to be colonized by cells and ultimately regenerated as largebone defects. Indeed, no adequate solutions have been found to date tosolve this clinical need.

For many decades, hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) has been recognizedas the prime material for bone scaffolding, as it closely resembles thecomposition of bone mineral and has demonstrated excellentbiocompatibility and osteoconductivity. However, the biomimicry ofhydroxyapatite is related to its nanosize and the presence of multipleions, partially replacing calcium and phosphate in the apatite lattice,which are the source of the biological activity of the bone during newtissue formation, remodeling and resorption.

The application of the sintering treatment to hydroxyapatite scaffoldsactivates surface and bulk reactions at the interface between adjacenthydroxyapatite grains that yield crystal ordering, with expulsion offoreign ions from the apatite lattice, and grain coalescence up toseveral micrometers, with reduction of specific surface, hydrophilicityand affinity with proteins and cells.

The extensive grain coalescence activated by the sintering processyields consolidation of the whole hydroxyapatite body through reductionof the intergranular porosity and, in turn, of the overall volume. Thisalso generates residual stresses which are among the main sources ofstructural defects in the ceramic materials. Indeed, the accommodationof residual stresses in ceramic materials is difficult due to their highrigidity (compared to metals and polymers), and is among the mostsignificant factors impairing the mechanical performance of ceramicmaterials, particularly in the case of large pieces characterized bycomplex shapes and porous structures, where volume variations followingheating/cooling cycles easily provoke critical structural damage.

For the above reasons, the classical ceramic synthesis process does notallow ceramic materials, in particular hydroxyapatites having abiomimetic composition and structure, high bioactivity andresorbability, to be manufactured. This is especially true when largeporous 3D ceramics are synthesized for the regeneration of critical sizebone defects (i.e. ≥2 cm).

Biomimetic composition and structure are of pivotal relevance forinducing the regenerative cascade in vivo that can uniquely determineand promote regeneration of large, load-bearing bone parts such as thelong bones of the limbs. These phenomena, which are closelyinter-related and must occur in synergy to activate and sustain theregeneration of bone with all its functions, are: i) fast osteogenesis,osteoconduction and osteointegration; ii) extensive blood vesselformation; iii) ability of progressive bio-resorption.

Fast osteogenesis and osteoconduction enable extensive bone formationand penetration into the scaffold, thus resulting in tight bone/scaffoldinterface and optimal osteointegration. To achieve these effects,bone-like chemical composition as well as wide open and interconnectedporosity are required, so that besides extensive penetration of new bonetissue, a simultaneous formation of a vascular network assisting theformation and maturation of the new bone can be achieved. Incompletecolonization of the scaffold may result in the formation of voids,fibrous tissues or necrotic areas, and will reduce the overall strengthand biomechanical performance of the bone/scaffold construct.

Within times compatible with new bone formation, the scaffold should beprogressively resorbed, to achieve optimal regeneration of the bonefollowing damage or disease. All the 3D bone scaffolds developed so farare based on sintered calcium phosphates that are crystalline materialshampering osteoclast activity, compared to nanocrystalline, nanosized,ion-substituted apatite; therefore, even though porous bonehydroxyapatite scaffolds can be well integrated into the surroundingbone by surface adhesion, the lack of bio-resorption does not allow thecomplete remodeling process, i.e. replacement of the scaffold with thenew bone. This results in incomplete recovery of the functional abilityof the diseased bone, particularly in the case of very long,load-bearing, bone segments.

Particularly in the case of long, load-bearing bones, the scaffold mustalso exhibit adequate mechanical performance, while maintaining wideopen macro-porosity, which is a challenge considering that thesefeatures are normally inversely related (i.e. the higher the porosity,the lower the mechanical resistance) and that a high porosity extent isrequired to provide adequate scaffold colonization and osteointegration.This is one of the most relevant factors limiting the application ofcurrent scaffolds in the regeneration of extensive portions of long,load-bearing bones. In this respect, scaffolds withhierarchically-organized porous structures can exhibit superiormechanical performance compared to materials with similar, but randomlyorganized porosity. In this respect, only scaffolds with such anorganized structure can efficiently activate mechano-transductionprocesses at the cell level, thus triggering regeneration of mature,organized and mechanically-competent bone.

The proposed innovation is based on a paradigmatic change from theclassical ceramic synthesis process to a new fashion of reactivesintering that enables the generation of ceramic phases with definedchemical composition, organized into a large 3D body with complexmorphology, hierarchical structure and, at the same time, optimizedmechanical performance, starting from hierarchically organized naturalstructures. In this respect biomorphic transformation is the fulcrum ofthis innovative approach that can be applied to hierarchically organizednatural structures (e.g. woods, plants, exoskeletons).

Biomorphic transformation of ligneous structures to bone-mimickingceramics was successfully attempted using woods with porous structuressuch as pinewood and rattan, and denser woods such as red oak and sipo,as templates for reproducing the structure and mechanical performance ofspongy and cortical bone, respectively. The use of wood in the formationof biomimetic hydroxyapatite scaffolds was reported by Anna Tampieri etal. in the Journal of Material Chemistry, 2009, 19, 4973-4980. In thispublication, Tampieri et al. describe the process of converting 1 cmlong pieces (therefore a small piece, not adequate for regeneration ofcritical size defects) of rattan wood and pine wood into hydroxyapatite.The process involved pyrolysis of the wood specimens at a temperature of1000° C. using a slow heating rate, followed by carburization whereinthe carbon template was transformed into calcium carbide. Carburizationwas achieved by either liquid phase infiltration or vapour phaseinfiltration. Vapour infiltration was performed at temperatures higherthan the boiling point of calcium (1484° C.). The carburization processinvolved initial heating the pyrolised wood to 800° C., followed byheating to 1100° C. and finally to 1650° C. for 3 hours. It wasnecessary to heat the pyrolised wood to this temperature for 3 hours toensure that the reaction went to completion. Following carburization,the three dimensional calcium carbide scaffold was oxidized to transformthe calcium carbide to calcium oxide, while preserving the morphology ofthe native wood. After oxidation, the three dimensional calcium oxidescaffold was carbonated to transform the calcium oxide scaffold intocalcium carbonate scaffold. High pressure values (2.2 MPa) were employedto allow the penetration of CO₂ across the forming CaCO₃ scale, up tothe core of the CaO structure. Finally, a phosphatization step wascarried out to transform the calcium carbonate scaffold intohydroxyapatite scaffold with hierarchically organized anisotropicmorphology resembling that of the native wood. During this step, thewood-derived CaCO₃ templates were soaked in an aqueous solution ofKH₂PO₄ at a temperature of 200° C., under a pressure of 1.2 MPa for 24hours.

The process described above yielded hydroxyapatite ceramic scaffoldswith the hierarchically organized anisotropic morphology of native wood.

The compressive strength of the scaffold derived from pinewood, measuredin the longitudinal direction ranged between 2.5 and 4 MPa, and in thetransversal direction, ranged between 0.5 and 1 MPa. Therefore onlyscaffolds of limited dimension, typically of less than 1 cm, areobtainable by said process. The low values of compression strength, alsoin association with a size ≤1 cm, make these scaffolds not relevant forbone regeneration, particularly in the case of load-bearing bones. Infact, it is accepted that, to be critical, a bone defect should have alength of 2-3 times the diameter of the affected bone. Hence, scaffoldof 1 cm in size cannot be considered as useful in this respect.

The phosphatization step mentioned above in the conversion of wood tohydroxyapatite, was reported in more detail by Ruffini et al. inChemical Engineering Journal 217 (2013) 150-158. In this publication,cylindrical templates of rattan-derived calcium carbonate havingdiameters of 8 mm and lengths of 10 mm were used as starting materials.The phosphatization process was carried out using aqueous solutions ofdiammonium hydrogen phosphate, ammonium dihydrogen phosphate andammonia.

Patent application WO 2012/063201 published on 18 May 2012, describes abone substitute comprising a core, based on hydroxyapatite, obtainedfrom at least one porous wood, and a shell based on hydroxyapatite orsilicon carbide obtained from at least one wood having a lower porositythan the at least one wood of the core. The shell was prepared in ahollow cylindrical shape suitable for accommodating the core, whichcould be prepared as a solid cylinder that is inserted into the cavityof the shell. The process for obtaining the bone substitute from wood isalso described in the application. The first step is pyrolysis of anative wood such as rattan or pine, by heating it to a temperature ofbetween 800 and 2000° C. From this process, a carbon material isobtained. In the second step, the carbon material is transformed intocalcium carbide at a temperature of between 1500 to 1700° C. Next, thecalcium carbide is oxidized at a temperature between 900 and 1000° C. Inorder to convert the calcium oxide material to calcium carbonate,carbonation is performed in an autoclave at a temperature of 400° C.with a CO₂ pressure of 2.2 MPa for 24 hours. The calcium carbonatematerial is then transformed into hydroxyapatite partially substitutedwith carbonate by phosphatization. The resulting hydroxyapatitescaffolds derived from rattan, have a compressive strength of between 4and 5 MPa in the longitudinal direction, and a compressive strength of 1MPa in the transversal direction.

Although the publications mentioned above describe the successfultransformation of wood such as rattan and pine into hydroxyapatite,while fairly reproducing the three-dimensional morphology of the wood,scaffolds exhibiting features adequate for regeneration of long segmentsof load-bearing bone could not be obtained.

Indeed all of the mentioned publications refer to hydroxyapatitescaffolds obtained from wood, having small dimensions (i.e. a volume ofless than 1 cm³) that cannot have real clinical applications,particularly for the regeneration of large, load-bearing bone parts. Theprocesses described in the prior art are not suitable for manufacturinghydroxyapatite scaffolds having dimensions that are convenient forclinical applications, such as for the regeneration of critical sizeload-bearing bone defects where large scaffolds, i.e. with size at leastequal to 2 cm, are needed.

Thus there remains a need in the art for a biomorphic scaffold, inparticular a porous 3D scaffold, with a biomimetic chemical compositionthat exhibits adequate mechanical performance, a morphology that isfavorable to cell colonization and vascular growth and, at the sametime, which has dimensions that are suitable for clinical applications.

Such a biomorphic scaffold would be particularly suitable for boneregeneration, in particular for implantation in load-bearing bonedefects, such as long bones of the limbs (e.g. femur, tibia, humerus,fibula, radius), but also for the substitution and regeneration of spinebones (e.g. vertebral bodies, intervertebral disc), cranial bone-partsor maxillofacial bone-parts.

The present disclosure meets the above needs by providing a biomorphicscaffold, preferably a hydroxyapatite scaffold particularly suitable forbone substitution and regeneration, in particular for substitution andregeneration of long load-bearing bones.

The present disclosure meets the above needs also by providing a processfor the manufacturing of a biomorphic scaffold, preferably a 3Dbiomorphic scaffold. In particular, the biomorphic scaffold is ahydroxyapatite scaffold.

SUMMARY OF THE DISCLOSURE

In general, the present disclosure describes a hydroxyapatite scaffoldobtained from a wood having a total porosity of between 60% and 95%,said porosity being measured after subjecting the wood to a step ofpyrolysis, the scaffold having a length, measured along a direction inwhich a dimension of the scaffold is maximum, greater than or equal to 2cm.

More particularly the present disclosure describes a biomorphichydroxyapatite scaffold obtained from a wood having a total porosity ofbetween 60% and 95% (said porosity being measured after subjecting thewood to a step of pyrolysis), said hydroxyapatite being characterized bya hierarchically organized pore structure and a compressive strength ofgreater than 5 MPa, preferably between 10 MPa and 20 MPa, measured inthe direction along the channel-like pores (longitudinal direction).

Preferably the hydroxyapatite of the disclosure shows a compressivestrength along the perpendicular direction of the long axis of thechannels (transversal direction) of up to 10 MPa.

Preferably, the biomorphic hydroxyapatite scaffold obtained from wood,has a hierarchically organized pore structure that derives from thehierarchically organized pore structure of the wood from which it isobtained (native wood).

The present disclosure also refers to a biomorphic hydroxyapatitescaffold derived from wood, wherein the hydroxyapatite is partiallysubstituted (doped) with one or more ions selected from the groupcomprising magnesium, strontium, silicon, titanium, carbonate, sodium,potassium, gallium, silver, copper, iron, zinc, manganese, europium andgadolinium.

The wood from which the biomorphic hydroxyapatite is derived has a totalporosity of between 60% and 95%, preferably between 65% and 85% (saidporosity being measured after subjecting the wood to a step ofpyrolysis). Woods exhibiting porosity within these ranges includerattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnutwood. Preferably the biomorphic hydroxyapatite is obtained from rattanwood.

The biomorphic hydroxyapatite scaffold obtained from wood of thedisclosure has structural cohesion and mechanical properties whichrender it particularly suitable for use as a bone substitute.

Therefore, the present disclosure refers to the use of the biomorphichydroxyapatite scaffold as bone substitute, as well as to a bonesubstitute comprising said biomorphic hydroxyapatite scaffold. Thedisclosure refers also to a bone substitute consisting of saidbiomorphic hydroxyapatite scaffold.

The biomorphic hydroxyapatite scaffold derived from wood may be used asa bone substitute for regenerating a bone or a bone portion, inparticular human and animal bones. Preferably, the biomorphichydroxyapatite scaffold derived from wood may be used as a bonesubstitute for bones or bone portions which are subjected to mechanicalloads. More preferably the bone or bone portions are long bones of theleg and arm such as the tibia, fibula, femur, humerus and radius.

The biomorphic hydroxyapatite scaffold derived from wood may also beused in the substitution and/or reconstruction of cranial bone-parts,maxillofacial bone-parts and spine bones (e.g. vertebral bodies,intervertebral disc).

The biomorphic hydroxyapatite scaffold of the disclosure can also beused as a filter for liquids or gases.

The present disclosure also describes a process for obtaining abiomorphic hydroxyapatite scaffold derived from wood.

More particularly the present disclosure describes a process forproducing a biomorphic hydroxyapatite scaffold having a length, measuredalong a direction in which a dimension of the scaffold is maximum,greater than or equal to 2 cm, which comprises the steps of pyrolysis,carburization, oxidation, hydration, carbonation and phosphatization ofa piece of wood (native wood) having a total porosity of at least 20%,preferably at least 40%, more preferably comprised between 60% and 95%,wherein said porosity is measured after subjecting the wood to the stepof pyrolysis. Examples of native wood that can be subjected to theprocess of the invention are: rattan, pine, abachi, balsa, sipo, oak,rosewood, kempas and walnut.

While multiple embodiments are disclosed, still other embodiments willbecome apparent to those skilled in the art from the following detaileddescription. As will be apparent, certain embodiments, as disclosedherein, are capable of modifications in various obvious aspects, allwithout departing from the disclosure. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE FIGURES

The following figures illustrate preferred embodiments of the subjectmatter disclosed herein. The claimed subject matter may be understood byreference to the following description taken in conjunction with theaccompanying figures, in which:

FIG. 1 shows a particular embodiment of the biomorphic scaffold thatfeatures a central channel.

FIG. 2 shows photographs depicting tissues of explanted mice calvariaafter 8 weeks. Extensive bone formation and penetration of the scaffoldpores was achieved to a similar extent when implanted alone or withosteogenic stromal cells, where a) represents wood and no cells, b)represents hydroxyapatite and no cells, c) represents wood and cells,and d) represents hydroxyapatite and cells. The arrows in FIG. 2 f)point to the channel-like pores of the scaffold which mimic Haversiansystems. Haversian systems or osteons are the functional unit of compactbone, in the form of cylinders made of bone lamellae organized inconcentric layers. In the middle of the osteons a channel exist,containing the bone's nerve and the blood supply.

FIG. 3 shows a stress-strain curve of a biomorphic scaffold subjected tocompressive loading, wherein y=stress (N), and x=strain (mm).

FIG. 4 shows the distribution of internal pore volume of the biomorphichydroxyapatite of the present disclosure compared with the pore volumeof the hierarchically structured hydroxyapatite obtained with a methodknown in the art.

FIG. 5 shows a graph depicting the enhanced viability of mesenchymalstem cells when in contact with scaffolds comprising 2 and 5 mol % ofSr, in comparison with a strontium-free scaffold (BC) after 24 hours, 48hours, 72 hours, 7 days and 14 days. y=% respect to BC.

FIG. 6 shows graphs depicting the expression of osteogenesis-relevantgenes, such as (a) RUNX2 and (b) ALP in scaffolds containing 2 mol %(Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium. y=fold-change expressionrelative to BC and x=days.

FIG. 7 shows a graph depicting osteoblast viability when in contact withscaffolds comprising 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of Sr, incomparison with a strontium-free scaffold (BC) after 24 hours, 48 hours,72 hours, 7 days and 14 days. y=% respect to BC.

FIG. 8 shows graphs depicting the expression of osteogenesis-relevantgenes, such as (a) Osterix, (b) BGlap, and c) IBSP in scaffoldscontaining 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium.y=fold-change expression relative to CT and x=days.

FIG. 9 shows graphs depicting the expression of osteoclast-relevantgenes, such as (a) Osacr, (b) CTSK, and c) Itg β3 in scaffoldscontaining 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium.y=fold-change expression relative to CT and x=days. A significantdecrease of the genes involved in the principal molecular pathways ofosteoclasts over time can be seen; thus indicating that the presence ofSr²⁺ ions in the scaffold inhibits osteoclast formation and activity; infigure a) the 14 day data were below the detection limit.

FIG. 10 shows a comparison of the pore distribution of two calciumcarbide scaffolds obtained after the carburization step of the prior artand the carburization step of the present invention, further comparedwith the pore distribution of the starting pyrolized wood. The specificsurface area of the two calcium carbide scaffolds are also reported inthe figure;

FIGS. 11, 12 and 13-top two pictures show SEM immages of two calciumcarbide scaffolds obtained with the process of the invention and theprior art process, respectively;

FIG. 13-bottom two pictures show a comparison of the dimension of thecarcium carbide granes;

FIG. 14 show a comparison of the crystal phase of the two calciumcarbide scaffolds, measured with x ray-XRD, obtained with the process ofthe invention and the prior art process, respectively.

FIG. 15 shows SEM immages of the calcium oxide scaffold obtained afterthe oxidation step of the present invention and the prior art oxidationstep;

FIG. 16 depicts the pore distribution of the two calcium oxide scaffoldsobtained after the oxidation step of the present invention and the priorart oxidation step, respectively;

FIGS. 17 and 18 show SEM immages of the calcium carbonate obtained afterthe carbonation step according to the present invention and the priorart carbonation step, respectively;

FIG. 19 shows that the pore distribution of the two calcium carbonatescaffolds obtained after the carbonation step according to the presentinvention and the prior art carbonation step, respectively;

FIG. 20 shows the comparison between the pore distribution of the finalbiomorphic hydroxyapatite scaffold (after phosphatization) obtained withthe process of the disclosure and the final biomorphic hydroxyapatitescaffold of the prior art;

FIGS. 21 shows the result obtained after subjecting a piece of rattanwood having a length, measured along a direction in which a dimension ofthe scaffold is maximum, equal or greater than 2 cm, to the processsteps according to the conditions described in the prior art: evenbefore the phosphatization step the scaffold can break down;

FIG. 22 shows the result obtained after subjecting a piece of rattanwood having a length, measured along a direction in which a dimension ofthe scaffold is maximum, equal or greater than 2 cm, to the processsteps according to the conditions described in the prior art: even ifthe scaffold survives the process steps up to phosphatization, afterphosphatization the scaffold breaks down;

FIG. 23 shows the relative quantification (2^(−ΔΔCt)) of gene expressionwith respect to the expression of the not-doped prior art scaffold usedas calibrator, after 14 days of mMSCs 3D cultured in dynamic conditionwith all the tested samples;

FIG. 24 shows an embodiment of the biomorphic scaffold with cuboidshape.

FIG. 25 shows that the channel-like structure of the biomorphichydroxyapatite scaffold obtained with the process of the presentdisclosure is uniquely characterized by pervious large channels (100-300micron in diameter) (micro CT Scan). Such channels are permissive to theformation of suitable blood vessels supporting bone regeneration.

DETAILED DESCRIPTION OF THE DISCLOSURE

As used in present description and in the appended claims, “hierarchicalpore structure” or “hierarchically organized pore structure” indicates amaterial having an anisotropic three dimensional pore structure in whichchannel-like pores are interconnected through transversal channels andwherein micro and nano-pores are present in the area surrounding thechannel-like pores.

As used in the present description and the appended claims, the valuesof “compressive strength” are obtained with the method described in thefollowing description by exerting mechanical forces along thelongitudinal and transversal directions, with respect to thechannel-like pores.

As used in the present description and the appended claims, “bonedefect” refers to a missing part or portion of the bone or to the entirebone that is missing and needs to be totally replaced by the scaffold ofthe disclosure.

As used in the present description, “biomorphic hydroxyapatite” refersto a material that: 1) consists of hydroxyapatite or 2) compriseshydroxyapatite or 3) is a material comprising or consisting ofhydroxyapatite and tricalcium phosphate. In case the material consistsof hydroxyapatite and tricalcium phosphate, the material is a biphasicmaterial. In an embodiment of the invention, when the “biomorphichydroxyapatite” is a material comprising or consisting of hydroxyapatiteand tricalcium phosphate, the hydroxyapatite is doped with one or moreions chosen in the group consisting of magnesium, strontium, silicon,titanium, carbonate, potassium, sodium, silver, gallium, copper, iron,zinc, manganese, europium, gadolinium and mixtures thereof.

The inventors of the present patent application have surprisingly foundthat it is possible to obtain a biomorphic hydroxyapatite scaffold fromwood which exhibits a biomimetic chemical composition, an adequatemechanical performance, a morphology that is favorable to cellcolonization and vascular growth and, at the same time, has dimensionsthat are suitable for clinical applications.

In a first aspect, the present disclosure describes a biomorphichydroxyapatite scaffold obtained from a wood having a total porosity ofat least 20%, preferably at least 40%, more preferably comprised between60% and 95%, wherein said porosity is measured after subjecting the woodto the step of pyrolysis , the scaffold having a length, measured alonga direction in which a dimension of the scaffold is maximum, greaterthan or equal to 2 cm.

The total porosity of the biomorphic hydroxyapatite scaffold obtainedafter the process of the disclosure is the same as the total porosity ofthe starting wood measured after subjecting the wood to the step ofpyrolysis. In particular, the total porosity of the biomorphichydroxyapatite scaffold obtained after the process of the disclosure isat least 20%, preferably at least 40%, more preferably comprised between60% and 95%.

Preferably, the scaffold has a length, measured along a direction inwhich a dimension of the scaffold is maximum, that is greater than orequal to 2 cm and reaches an end value that is determined according tothe clinical application. For example in the case of bone substitutionof long bones, such as tibia, femur, fibula, humerus, radius, the lengthof the scaffold, measured along a direction in which a dimension of thescaffold is maximum, can be comprised between 2 and 20 cm.

Preferably, the scaffold of the disclosure has a compressive strengthmeasured in the longitudinal direction of greater than 5 MPa, preferablybetween 10 MPa and 20 MPa.

Preferably the scaffold of the disclosure shows a compressive strengthalong the transversal direction of up to 10 MPa.

Preferably, the biomorphic hydroxyapatite scaffold is characterized by ahierarchically organized pore structure.

The “hierarchical pore structure” or “hierarchically organized porestructure” of the hydroxyapatite scaffold of the disclosure derives fromthe complex three-dimensional hierarchical structure of the startingwood from which the scaffold is obtained and therefore has a range ofdifferently sized pores. The differently sized pores in the hierarchicalstructure render it desirable for use as a bone substitute.

For example, pores having diameter ≥200 μm, preferably between 150-300μm, more preferably 200-300 μm will permit cell colonization andproliferation and the formation of an appropriate vascularization tree.Pores having a diameter ≤10 μm, preferably <1 μm, more preferablybetween 0.01 and 0.1 μm (micro and nano-pores), that partiallyinterconnect the channel-like pores, permit exchange of nutrient fluidsand discharge of the waste products of cell metabolism.

The preservation of the hierarchical pore structure of wood in thehydroxyapatite, provides scaffolds with optimal mechanical features andenables the efficient discharge of mechanical loads.

As hydroxyapatite obtained from wood reproduces the structure of anatural material in detail, it can be thus referred to as beingbiomorphic.

In particular, the hierarchically organized pore structure of thescaffold of the disclosure includes between 30% and 80% (of the totalporosity) of pores having a diameter below 150 μm, the reminder to 100%of total porosity being pores having diameter greater than 150 μm.

In one embodiment, preferably when the starting wood is rattan, between30% and 60% of the total porosity of the scaffold is due to pores havinga diameter ≤10 μm

In one embodiment, preferably when the starting wood is rattan, at least25% of the total porosity, preferably between 25% and 50% of the totalporosity of the hydroxyapatite scaffold is due to pores having adiameter ≤1 μm, preferably ≤0.1 μm, in particular between 0.01 and 0.1μm.

In one embodiment, preferably when the starting wood is rattan, at least20% of the total porosity of the hydroxyapatite scaffold is due to poresthat have diameter 150 μm.

Preferably, the hydroxyapatite scaffold has a specific surface area(SSA)>9 m²/g, preferably from 9 to 20 m²/g.

The wood used to obtain the hydroxyapatite scaffold can be any woodhaving a total porosity of at least 20%, preferably at least 40%, morepreferably comprised between 60% and 95%, even more preferably aporosity of between 65% and 85% (said porosity being measured aftersubjecting the wood to a step of pyrolysis).

Examples of suitable woods used to obtain the hydroxyapatite includerattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnutwood, preferably the wood used is rattan wood.

The hydroxyapatite scaffold obtained from wood may comprisehydroxyapatite which is partially substituted with one or more ions.Examples of such ions are carbonate, magnesium, strontium, silicon,titanium, sodium, potassium, silver, gallium, copper, iron, zinc,manganese, europium and gadolinium. The introduction of carbonate in thephosphate site increases bio-solubility and enhances surface affinity toosteoblast cells.

The introduction of magnesium provides enhanced ability of new boneapposition and formation. The introduction of strontium assists inre-establishing bone production, affected by metabolic diseases such asosteoporosis, so that its presence may enhance bone regeneration.

The introduction of silver, gallium, copper and zinc providesantibacterial properties. When the hydroxyapatite scaffold obtained fromwood comprises hydroxyapatite which is partially substituted with one ormore ions, the scaffold is a material comprising or consisting of dopedhydroxyapatite and tricalcium phosphate. According to a furtherembodiment, the biomorphic hydroxyapatite scaffold of the instantdisclosure may comprise:

0-15 wt % of magnesium, preferably 1-10 wt %; and/or

0-15 wt % of carbonate, preferably 1-10 wt %; and/or

0-15 wt % of strontium, preferably 1-10 wt %; and/or

0-20 wt % of titanium, preferably 1-10 wt %; and/or

0-15 wt % of potassium, preferably 1-10 wt %; and/or

0-15 wt % of sodium, preferably 1-10 wt %; and/or

0-15 wt % of silicon, preferably 1-10 wt % and/or;

0-15 wt % of silver, preferably 1-10 wt % and/or;

0-15 wt % of gallium, preferably 1-10 wt % and/or;

0-15 wt % of copper, preferably 1-10 wt % and/or;

0-30 wt % of iron, preferably 1-10 wt %; and/or

0-15 wt % of zinc, preferably 1-10 wt % and/or;

0-15 wt % of manganese, preferably 1-10 wt % and/or;

0-15 wt % of europium, preferably 1-10 wt % and/or;

0-15 wt % of gadolinium, preferably 1-10 wt % and/or.

The biomorphic hydroxyapatite scaffold obtained from wood according tothe present disclosure, has bioactivity and bioresorbabilitycharacteristics combined with mechanical strength characteristics anddimensions that makes it particularly suited for clinical use as a bonesubstitute, in particular in humans and animals. Such a bone substitutecould be used to substitute and/or reconstruct and/or regenerate bone,bone portions or bone defects. For example, the bone substitute could beused to substitute or regenerate bone or bone portions that aresubjected to mechanical loads. For example, the bone substitute could beused to substitute or regenerate long bones of the arms and legs. Suchlong bones could include the tibia, femur, fibula, humerus, radius, etc.

The bone substitute could also be used in the substitution and/orreconstruction of cranial bone-parts, maxillofacial bone-parts and spinebones e.g. vertebral bodies, intervertebral disc) and in spinal fusionsurgery procedures.

When used as a bone substitute, the biomorphic hydroxyapatite scaffoldcan have a shape that adapts to the shape of the bone defect that needsto be reconstructed in such a way as to substantially fill the bone gap.Therefore, the scaffold and the bone substitute of the disclosure canhave any shape that is suitable for the purpose of reconstructing andregenerating a bone defects or for substituting any missing part of thebone.

For example the scaffold or the bone substitute of the presentdisclosure may take the form of a cylinder, right prism, or cuboid, orwedges. In one embodiment, the scaffold or the bone substitute comprisesa central channel with a diameter of between about 20% to about 60% ofthe diameter of the scaffold or the bone substitute. In particular, thescaffold or the bone substitute has a tubular shape.

In one embodiment, the present disclosure further refers to a scaffoldor a bone substitute having a cylindrical, right prism, cuboid ortubular shape, having a height greater than or equal to 2 cm.

In an embodiment of the present disclosure, the scaffold or the bonesubstitute may be coated with a thin layer based on hydroxyapatiteand/or collagen to increase cellular adhesion and proliferation, andthus osteointegration in the surrounding bone tissue. The layer mayadditionally comprise hydroxyapatite substituted with one or more ionsrelevant for the stimulation of the bone regeneration such as carbonate,magnesium, silicon, potassium, sodium and strontium, or withantibacterial effect such as gallium, silver, copper or zinc.

In a further embodiment of the present disclosure, the scaffold or thebone substitute may be soaked in a natural polymer (chosen among thegroup comprising gelatin, collagen, alginate, chitosan, gellan,cellulose) to further increase mechanical properties and furtherpromoting cell adhesion.

To the scaffold or bone substitute cells, platelet rich plasma,antibodies, growth factors proteins, DNA fragments, miRNA, siRNa can beadded in order to help cell adhesion.

Also drugs, such as antibiotics or anticancer drugs, can be added to thescaffold or bone substitute.

The disclosure refers also to a method of reconstruction and/orregeneration of a human or animal bone having a bone defect, comprisingthe steps of:

providing a bone substitute comprising or consisting of the biomorphichydroxyapatite scaffold of the disclosure having a shape thatcorresponds to the shape of a bone defect;

implanting the bone substitute in the bone defect of the patient.

Preferably, the method of reconstruction and/or regeneration includesthe steps of providing a 3D model of the bone defect and, based on the3D model obtained, imparting to the scaffold a shape corresponding tothe shape of the bone defect. The step of imparting a shape to thescaffold can be applied to the starting piece of wood or to thehydroxyapatite scaffold obtained at the end of the transformationprocess of the disclosure or to the scaffold obtained after each step ofthe process (e.g. after the carbonation step). Preferably the step ofimparting the shape is applied on the starting piece of wood.

The biomorphic hydroxyapatite scaffold of the disclosure and thebiomorphic hydroxyapatite scaffold partially substituted with one ormore ions, is obtained from a multistep transformation processcomprising the following steps:

1) Pyrolysis: a native wood is heated at a temperature in the range of600° C. to 1000° C. under an inert atmosphere to permit thedecomposition and the elimination of all organic substances. From thisprocess, a carbon template is obtained.

2) Carburization: the carbon template is infiltrated with calcium in thevapour state at a temperature in the range 900 to 1200° C., and at apressure <1000 mbar, preferably <600 mbar, more preferably in the rangeof 0.05 to 100 mbar, thus transforming the carbon template into calciumcarbide (CaC₂).

3) Oxidation: the calcium carbide template is heated in air at atemperature in the range of 750 to 1300° C., preferably 1000-1200° C.,thus enabling the transformation of calcium carbide into calcium oxide(CaO).

4) Hydration: the calcium oxide template is exposed to water, thusenabling water uptake in an amount of 1-25 mole %, preferably 5-15 wt %.

5) Carbonation: the calcium oxide template is transformed into calciumcarbonate by heating at a temperature in the range of 500 to 900° C.,preferably at a temperature in the range of 750 to 850° C. under a CO₂pressure, or a mixture of CO₂ and an inert gas (e.g. argon, nitrogen)pressure. The pressure range is from 4 to 20 MPa.

6) Phosphatization: the calcium carbonate template is treated with atleast one phosphate salt.

In the pyrolysis step 1) of the multi-step process, the native wood ispreferably selected among rattan, pine, abachi, balsa sipo, oak,rosewood, kempas and walnut wood. More preferably the native wood israttan wood.

The native wood has a total porosity of at least 20%, preferably atleast 40%, more preferably comprised between 60% and 95%, wherein saidporosity is measured after subjecting the wood to the step of pyrolysis.

Prior to the pyrolysis step 1), the starting native wood may beoptionally dried at a temperature of between 50° C. and 90° C.,preferably at a temperature of between 60° C. and 80° C., morepreferably at a temperature of between 65° C. and 75° C. The native woodmay be dried for more than 6 hours, preferably for more than 12 hours,preferably for more than 18 hours, preferably for a time comprisedbetween 20 and 30 hours.

In the pyrolysis step of the multi-step process, the inert atmospheremay be an atmosphere of a gas selected from the group comprisingnitrogen and argon.

In the pyrolysis step of the multi-step process, the native wood may beheated at a temperature of between 600° C. to 1000° C., preferably at atemperature of between 800° C. and 1000° C. The pyrolysis step may lastmore than 6 hours, preferably more than 12 hours, preferably more than18 hours, preferably said step may last for a time comprised between 20and 30 hours.

The thermal cycle of the pyrolysis step 1) may be carried out by heatingthe native wood at the rate not higher than 5° C./min, preferably nothigher than 3° C./min and by cooling at a rate not higher than 3°C./min, preferably not higher than 2° C./min, to prevent crack formationand internal fracture of the material.

Prior to the pyrolysis step 1), the multistep method can additionallycomprise a step i) of selection and preparation of the native wood,wherein said native wood can be cut into a piece having a shapecorresponding to the shape of a bone defect to be reconstructed. Inparticular the native wood is shaped into a piece of wood having alength, measured along a direction in which a dimension of the wood ismaximum, that is greater than or equal to 2 cm. Preferably, thedimension of the wood reaches an end value that is determined accordingto the clinical application.

For example, the native wood can be shaped in the form of a cylinder,right prism, or cuboid. The native wood can also be shaped in such a wayas to comprise a central channel with a diameter of between about 20% toabout 60% of the diameter of the piece of wood. In particular, thenative wood can be cut into a tubular shape.

Preferably, step i) of selection and preparation of the native wood,comprises the steps of: providing a 3D model of a bone defect and, basedon the 3D model obtained, imparting to the native wood a shapecorresponding to the shape of the bone defect. The step of imparting ashape to the native wood can be applied to the starting native wood orto the hydroxyapatite scaffold obtained at the end of the multi-steptransformation process. Preferably the step of imparting the shape isapplied on the starting native wood to avoid internal and externaldamage (fracture) of the scaffold.

In the carburization step 2) of the multi-step process, the reaction ispreferably carried out with a Ca/C molar ratio (at the beginning of thereaction) in the range of 1.10 to 2.50, preferably in the range of 1.50to 2.00. The Ca/C molar ratio is important because ratios below therange lead to incomplete reactions and ratios above the range lead toobstruction of the pores by residues of Ca.

In the carburization step of the multi-step process, the carbon templateis heated at a heating rate in the range of 1 to 10° C./min, preferablyat a heating rate in the range of 1 to 7° C./min.

The inventors of the present patent application have surprisingly foundthat carrying out the carburization step at a reduced pressure as abovedescribed is an advantage for the successful application of thesubsequent process steps, particularly when large biomorphic scaffoldneeds to be produced.

In fact, by using the above-described pressure conditions, theevaporation of calcium can occur at temperatures that are about 400-500°C. lower than the boiling point of calcium at room atmosphere (i.e.1484° C.), thus yielding, unexpectedly, the complete transformation ofthe pyrolized wood into calcium carbide at a temperature much lower thanany other process known in the art. In particular, the use of pressurein the range of 0.5-600 mbar, or preferably 0.05 to 100 mbar, results ina substantially complete transformation of the pyrolized wood intocalcium carbide.

A substantially complete transformation of the pyrolized wood intocalcium carbide will result in an advantage for the yields of thesubsequent transformation steps.

The carburization conditions of the present disclosure also improvepreservation of the micro- and nano-pores having diameters ≤1 μm(preferably from 0.01 to 0.1 μm) of the native wood both in the scaffoldafter carburization and in the final biomorphic scaffold, with respectto the known scaffolds obtained from wood using known processes.

The carburization phase is a critical step in the process because a goodpreservation of the micro- and nano-porosity after this step will ensurethat the final biomorphic scaffold exhibits similar nano/micro-porosity.The presence of a high percentage of well interconnected micro andnano-pores in the final biomorphic scaffold permits exchange of nutrientfluids and discharge of the waste products of cell metabolism.

Besides improving the preservation of the micro- and nano-pores havingdiameters ≤1 μm, the carburization conditions here described yield ascaffold after carburization (and also a final biomorphic scaffold) withspecific surface area (SSA) from 9 to 20 m²/g. Such a specific surfacearea is about 2-fold higher than the SSA of a scaffold obtained with aprocess known in the art—which is about 5-6 m²/g (see comparativeexample 4 and FIGS. 10-13).

The carburization conditions of the present disclosure also yield aporous calcium carbide scaffold containing calcium carbide grains thatare smaller than the grains of a scaffold obtained with a known process(see comparative example 4 and FIG. 13-bottom pictures).

The comparative examples show that the dimensions of the calcium carbidegrains in the scaffold after carburization according to the presentdisclosure is about 5-15 μm (preferably about 10 μm), while the crystaldimensions of the scaffold after carburization obtained with knownprocesses is about 100 μm.

The inventors of the present patent application have surprisingly foundthat the higher specific surface area (SSA) and the smaller dimension ofthe grains that are obtained with the carburization conditionsabove-described, and could not obtained by previously disclosed methods,is important to ensure high yield of transformation of the native woodafter each step of the process.

Comparative example 4 and FIG. 14 also show that the scaffold aftercarburization according to the present disclosure contains calciumcarbide with a mixture of tetragonal and cubic crystal lattice, whilethe scaffold obtained with known processes contains calcium carbide witha tetragonal crystal lattice only. Since calcium oxide has a cubiccrystal structure only, the transformation from a calcium carbide, whichis partially in a cubic form, to calcium oxide, can occur with a lowerrisk of generating microfractures in the scaffold. This is extremelyadvantageous for the quality of the final biomorphic scaffold.

Therefore, the conditions employed in the carburization step stronglyreduce the number of defects that can be observed in the hierarchicalpore structure of the calcium carbide.

In addition, the low temperatures employed in the present disclosure(i.e. well below 1500° C.) prevent grain coalescence and excessiveconsolidation of the calcium carbide, which provoke structuraldistortion and deviations from the original microstructure of thestarting wood, thus impairing the outcome of the following processsteps.

In the oxidation step 3) of the multi-step process, the calcium carbidetemplate may be heated to a final temperature in the range of 800 to1300° C., preferably to a final temperature in the range of 1000 to1200° C.

In the oxidation step, the calcium carbide template may be heated at aheating rate in the range of 1 to 15° C./min, preferably at a heatingrate in the range of 1 to 7° C./min.

The oxidation of calcium carbide obtained under pressure according tothe carburization step described above leads to a scaffold of calciumoxide with higher specific surface area (SSA) and a porosity with ahigher micro- and nano-pores fraction with respect to scaffoldsobtainable with the known processes (see comparative example 4 and FIG.16). The comparative experiment show that the micro and nano-porosity isconserved also after the oxidation step.

In the hydration step 4) of the multi-step process the calcium oxidetemplate is exposed to water, thus enabling water uptake in an amountpreferably comprised in the range of 1-25 mole %, more preferablycomprised in the range 5-15 mole %. This step leads to the formation ofhydrated calcium oxide containing calcium hydroxide in amount 50% byweight of the 3D structure, that catalyzes the subsequent carbonation ofCaO. The amount of calcium hydroxide (Ca(OH)₂) as intermediate productmust be strictly controlled to avoid the collapse of the 3D structure.The hydration conditions here described allow to keep the amount ofcalcium hydroxide ≤50%.

In a preferred embodiment, the hydration step is conducted at the sametime as the carbonation step, for example by using CO₂ enriched withwater.

In the carbonation step 5) of the multi-step process, the use of hightemperature while progressively increasing the CO₂ pressure in thesystem up to the values indicated above, surprisingly enablessubstantially complete conversion of hydrated calcium oxide into acalcium carbonate template which exhibits surprisingly high cohesion andmechanical strength.

The carbonation step of the multi-step process may be carried outaccording to one of the following thermal cycles:

at a constant CO₂ pressure of about 10-15 MPa, slowly increasing thetemperature at a value in the range of about 750-850° C., preferably atabout 800° C.;

at a constant temperature of about 750-850° C. (or about 700-800° C.),preferably at about 800° C. raising the pressure up to about 10-15 MPa;

keeping the pressure at about 4-6 MPa while raising the temperature upto about 750-850° C. (or about 700-800° C.), preferably up to about 800°C. and subsequently increasing the pressure up to about 10-15 MPa.

The carbonation process occurs through the formation of reactiveintermediates, such as calcium hydroxide. This leads to a final calciumcarbonate characterized by a fine-grained structure substantially freeof any large cubic crystal of calcium carbonate (>10 μm), which mightcompromise the structural integrity of the 3D structure. Comparativeexample 4 and FIGS. 17-18 show that, thanks to the application of ahydration step, after the carbonation step a finer-grained structure isobtained, compared to the intermediate 3D structure that is obtainedwith the processes known in the art.

The carbonation step carried out in the conditions described aboveresults in superior mechanical properties of the biomorphichydroxyapatite scaffold obtainable by the process of the disclosure whencompared to similar processes known in the art, in which the carbonationstep is carried out at high temperature and low pressure or at highpressure and low temperature.

The inventors of the present patent application have surprisingly foundthat the achievement of the above reported features in the calciumcarbonate is an important condition to enable the completetransformation of large pieces (i.e. ≥2 cm) into a final biomorphicscaffold having the desired composition and maintenance of the originalwood microstructure.

In the phosphatization step 6) of the multi-step process, the at leastone phosphate salt may be selected from the group consisting of ammoniumphosphate, sodium phosphate, and potassium phosphate. The use ofammonium phosphate enables a better control of the pH, hence theconversion process is more efficient and the resulting body hasfavorable mechanical properties and physical cohesion.

In the phosphatization step of the multi-step process, the calciumcarbonate template may be immersed in a solution comprising at least oneof said phosphate salts. The solution may have a concentration of 0.1 to5M, preferably a concentration of 0.5 to 2.0M.

The starting ratio of PO₄/CO₃ in the phosphatization step of themulti-step process is preferably 1.5 to 5 times the theoreticalstoichiometric value, preferably 2 to 4 times the theoreticalstoichiometric value.

In the phosphatization step of the multi-step process, the calciumcarbonate template immersed in a phosphate-rich solution may be heatedfrom 25° C. to 300° C. under a vapour pressure in the range of 0.1 to2.5 MPa (hydrothermal conditions).

The phosphatization step may last about 12 to about 180 hours,preferably about 48 to about 120 hours, more preferably from 24 to 72hours.

The starting pH of the phosphate-rich solution in the phosphatizationstep of the multi-step method is preferably between pH 7 and 12.

Substitution of the hydroxyapatite with other ions can be achieved byintroducing suitable soluble salts containing the ions of interestduring or after the process completion, preferably during thephosphatization process. Suitable ions may include strontium, magnesium,silicon, titanium, carbonate, sodium, potassium, gallium, silver,copper, iron, zinc, manganese, europium, gadolinium, and mixturesthereof. An example of a solution containing magnesium ions isMgCl₂*6H₂O, and an example of a solution containing strontium ions isSrCl₂*6H₂O.

As a consequence of the ionic doping, the final biomorphic scaffoldcomprises or consists of a material comprising or consisting of dopedhydroxyapatite and tricalcium phosphate.

According to a less preferred embodiment, where the native wood has notbeen shaped into form and dimensions suitable for being used as bonesubstitute (i.e. if step i) is not carried out), the biomorphichydroxyapatite scaffold obtained from the multi-step process mayconveniently be shaped into a scaffold having the required form andshape by known techniques.

The disclosure relates also to the biomorphic hydroxyapatite scaffoldobtained (or obtainable) from the process described above, havingimproved physical and mechanical properties if compared tohierarchically structured hydroxyapatites obtained by similar processesknown in the art.

In particular, the biomorphic hydroxyapatite scaffold obtained (orobtainable) from the process of the present disclosure possess all thefeatures above described for the scaffold or bone substitute of thedisclosure.

In particular, with respect to the prior art, the final biomorphicscaffold obtained with the process here described possess a porositywhich is composed by a higher percentage of micro and nano-pores thanthe scaffolds obtained by similar processes known in the art. Inparticular at least 25% of the total porosity, preferably between 25%and 50% of the total porosity of the hydroxyapatite scaffold of thedisclosure is due to pores having a diameter ≤1 μm, preferably ≤0.1 μm,in particular between 0.01 and 0.1 μm.

This high percentage of micro and nano-porosity is extremelyadvantageous from a clinical point of view because micro and nano-porespermit exchange of nutrient fluids and discharge of the waste productsof cell metabolism enhance, thus improving bone regeneration.

Moreover, the biomorphic scaffold of the disclosure shows a higherspecific surface area (9 to 20 m²/g vs 5-6 m²/g) than a scaffoldobtained with a process known in the art. A higher surface areadetermines enhanced surface bioactivity and enhanced wettability of thescaffold or bone substitute, thus improving the osteointegration andbio-resorption process.

Also the biomorphic scaffold of the disclosure includes hydroxyapatitegrains of about 100-200 nm (i.e. nano-grains), much smaller than theones present in sintered hydroxyapatite (i.e. typically >1 μm). Smallgrains show a clinical advantage for bone regeneration because they canbe more easily resorbed by the cells, thus allowing a better boneregeneration with respect to the scaffold known in the art.

In addition, the biomorphic scaffold of the disclosure exhibitscompressive strength greater than 5 MPa, preferably between 10 MPa and20 MPa, measured in the direction along the channel-like pores(longitudinal direction), and a compressive strength along the directionof the transversal channels (transversal direction) of up to 10 MPa. Theinventors of the present disclosure surprisingly found that thesemechanical features make the final hydroxyapatite scaffold as astand-alone material, therefore it can be applied in procedures ofregeneration of load-bearing bone parts without the use of anyreinforcing or sustaining structure such as shells or bars.

In nature, nanocrystalline, ion-substituted hydroxyapatite is the maincomponent present in hard body tissues; in fact, the mineral phase inbone is a nanostructured phase composed of finely dispersedhydroxyapatite platelets of dimensions below 100 nm that organize in a3D hierarchically organized porous structure representing the whole bonetissue.

In this respect, the inventors of the present disclosure surprisinglyfound that, when compared with previously known art, the biomorphictransformation of natural wood structures obtained by the abovedescribed process, can uniquely give rise to final hydroxyapatite bonescaffolds exhibiting simultaneously bone-mimicking composition, highopen and interconnected macro/micro/nano-porosity and superiormechanical strength, associated with a size relevant for application inload-bearing sites, particularly in long segmental bones of the limbs,or large maxillofacial regions, or in spine.

All these features, which have never been shown to occur simultaneously,are of outmost importance to enable extensive bone regeneration inload-bearing sites. The differences in the biomorphic hydroxyapatitescaffold structure obtainable with the process according to the presentdisclosure yield important clinical advantages that are shown incomparative example 6. In particular, the present scaffolds show ahigher inductive power on the expression of osteogenic related genes,with respect to the prior art scaffolds, which translate in a betterclinical performance in terms of bone regeneration.

The present disclosure is further illustrated by the following,non-limitative, examples.

EXAMPLES

Methods of Measurement

Total porosity of wood subjected to a pyrolysis step (pyrolized wood): apiece of pyrolyzed wood, shaped as a prism or a cylinder, is weighed,then the volume is obtained by measuring diameter and height. Theabsolute density (A.D.) of the pyrolyzed wood is obtained byweight/volume ratio; the relative density (R.D.) is obtained by dividingthe A.D. of the pyrolyzed wood by the theoretical density of carbon(i.e. R.D.=A.D./2.25). The total porosity (%) is obtained by(1-R.D)*100.

Total porosity of a scaffold obtained after each step of the process andof the biomorphic hydroxyapatite scaffold obtained at the end of theprocess:

The porosity is calculated by applying the same method as above usingappropriate values for the theoretical density of each material obtainedafter each step (i.e. the theoretical density of CaC₂, CaO, CaCO₃, HA).

Compressive strength: the final scaffold or bone substitute, shaped as aprism or a cylinder, is subjected to loading by using a universalscrew-type testing machine to obtain stress-strain curves and thefracture load. The compressive strength is given by the ratio betweenthe fracture load and the area subjected to compression.

Pore diameter: the pore size distribution and pore morphology of thefinal scaffold or after each step of the process are evaluated by meansof mercury intrusion porosimetry and scanning electron microscopy (SEM),respectively. Mercury intrusion porosimetry analysis is based on themeasure of the intrusion of mercury into the pores of the sample atvarious pressures.

Crystalline phases: identification and quantification: of thecrystalline phases on scaffolds are performed by X-ray powderdiffraction technique (XRD), evaluating the result of the X-radiationincidence on the sample, with different and continuous angles.

Specific Surface Area: the total surface area of the materials per unitof bulk volume (m²/g) is evaluated using the BET method, estimated fromthe amount of gas adsorbed in relationship with its pressure.

Example 1

Preparation of a Hydroxyapatite Derived from Wood Using the Multi-StepProcess:

i) A piece of native rattan wood is shaped in a cylindrical form havingthe following dimensions: diameter=2 cm; height=3 cm;

1) Pyrolysis of the Native Wood

The starting wood piece is dried at 70° C. for 24 hours, then treated at800° C. for more than 30 minutes under flowing nitrogen, thustransforming into pure carbon template. Thermal cycle: heating at 1°C./min up to 350° C. and 2° C./min from 350° to 800° C. The sample ismaintained at temperature of 800° C. for about at least 30 min andsubsequently the template is cooled at 1° C./min.

2) Carburization

The carbon template is subjected to heating at 1000° C. under argon andcalcium atmosphere at 0.5 mbar, thus transforming in calcium carbide.Dwell time at 1000° C.=30 minutes.

3) Oxidation

The calcium carbide template is heated in air up to 1100° C. following aheating rate in the range of 1-7° C./min, thus enabling the completetransformation into calcium oxide.

4) Hydration

The calcium oxide body is activated by exposure to water, thus enablingwater uptake in the amounts of about 10 mole %.

5) Carbonation

The pre-conditioned hydrated body is heated to 800° C. under aprogressively increasing CO₂ pressure of 0.5 to 10 MPa. This transformsthe calcium oxide body into calcium carbonate.

6) Phosphatization

The calcium carbonate body is immersed in a 0.5 M ammonium phosphatesolution and a starting PO₄ to CO₃ ratio of 2 times the theoreticalstoichiometric value, at a temperatures of 200° C. under a water vaporpressure of 2 MPa.

The compressive strength of the scaffold was evaluated by exertingmechanical forces along the perpendicular and transversal direction,with respect to the orientation of the channel-like pores.

By loading along the pore direction (which is the mostclinically-reflective configuration to mimic the in vivo biomechanicalstimuli in the case of long segmental bones), the scaffold (developed asa hollow cylinder with outer diameter=15 mm; inner diameter=6 mmheight=20 mm, and a pore extent of 60-65 vol %) exhibited compressivestrength of up to 16 MPa (i.e. 250 Kg of ultimate load (FIGS. 1 and 3).In the transversal direction, the scaffold exhibited compressivestrength of up to 4 MPa.

The scaffold could also be subjected to thermal treatment at a maximumtemperature of 1300° C., in a controlled atmosphere, to further increasethe mechanical strength of the scaffold.

The bone-like microstructural features of the biomorphic scaffoldenables delivery of topological information to cells to build new bonetissue with organized structure. This was confirmed by in vivo testswhere the scaffold was implanted in rabbit femurs and mouse calvaria.

The scaffold did not induce any toxic adverse reactions nor any necrosisor infections after surgery. The scaffold yielded extensive colonizationby the newly formed bone after 1 month, similar to the control which wasa commercial porous apatite scaffold: EngiPore, Finceramica S.p.A.,Italy.

The tissues explanted from mice calvaria showed extensive bone formationand penetration into the scaffold pores both when the scaffold wasimplanted alone and also when osteogenic stromal cells were added to theimplanted scaffold (FIG. 2a-d ). The channel-like porosity of thescaffold induced the formation of bone structures mimicking Haversiansystems (as indicated by the arrows in FIG. 2f ). Moreover, thechannel-like pores of the scaffold induced fast angiogenesis so toassist the formation and penetration of the new bone. This resultconfirms that a suitable orientation of the porosity in relationship tothe orientation of the endogenous vascular network can be effective inpromoting early development of extensive angiogenesis.

Example 2

Comparison of Biomorphic Hydroxyapatite of the Disclosure andHierarchically Structured Hydroxyapatite Known in the Art

A comparison test was made between the pore size distribution ofbiomorphic hydroxyapatite of the present disclosure obtained from rattannative wood and the pore size distribution of a hierarchicallystructured hydroxyapatite obtained from the same native wood accordingto the teaching of Anna Tampieri et al. in the Journal of MaterialChemistry, 2009, 19, 4973-4980 using the teaching of Ruffini et al. inChemical Engineering Journal 217 (2013) 150-158 for the phosphatizationstep only (Mixture of NH₄H₂PO₄—(NH₄)₂HPO₄, pH=9, Tmax=60° C., time=80h).

The results are shown in FIG. 4, wherein the black columns refer to thebiomorphic hydroxyapatite of the disclosure and the dark gray columns tothe hydroxyapatite known in the art.

It is evident the increasing in the number of pores having diametercomprised in the range 200-300 μm in the biomorphic hydroxyapatite ofthe present disclosure in comparison with known hierarchically organizedhydroxyapatite, said pores being the ones with the most appropriatedimensions to promote a physiological vascularization of the biomorphichydroxyapatite when implanted as bone substitute.

Moreover, the same FIG. 4 shows an increasing number of pores havingdiameter in the range 0.01-0.1 micron, which clearly indicates that themicrostructure of the native wood is preserved in the final product.

Example 3

Preparation of a hydroxyapatite derived from wood doped with Mg²⁺ and/orSr²⁺:

Steps 1 to 5 of the multi-step method as described in example 1 arefollowed to yield the calcium carbonate body. Doping with Mg²⁺ and/orSr²⁺ ions has been achieved according to each of the following methods:

Method 1

A solution of Sr²⁺ (in the form of SrCl) is added to a 1.0 Mphosphate-rich solution. The calcium carbonate body prepared accordingto the multi-step method is then immersed in the combined solution andheated to a temperature of 200° C. under a water vapor pressure of 2MPa. This yields a Sr²-doped hydroxyapatite with the morphology of thestarting wood piece.

Method 2

The calcium carbonate body is immersed in 1.0 M phosphate-rich solution.Whilst heating to a temperature of 25-90° C. under a water vaporpressure of 0.1 MPa, a solution Sr²⁺ is progressively added. This yieldsa Sr²-doped hydroxyapatite with the morphology of the starting woodpiece.

Method 3

The pure calcium carbonate body (or partially converted inhydroxyapatite by immersion in 1.5 M phosphate-rich solution at roomtemperature or higher for 24 h) is immersed in an aqueous or organicsolution containing Sr²⁺ ions for 24 h. It is then removed from thesolution and is immersed in 1.5 M phosphate-rich solution. Whilstheating to a temperature of 200° C. under a water vapor pressure in therange 0.5-1.5 MPa. This yields a Sr²-doped hydroxyapatite with themorphology of the starting wood piece.

Properties of the Scaffold Substituted with Strontium

Hydroxyapatite scaffolds substituted with strontium were developed andwere found to exhibit enhanced viability of mesenchymal stem cells(MSCs) when compared to the strontium-free scaffolds (FIGS. 5).

The strontium scaffolds also displayed well-spread morphology andincreased expression of osteogenesis-relevant genes, such as RUNX2 andALP (FIG. 6), thus acting as promoters of osteoblastic differentiation.In particular, when compared to the Sr-free scaffold, a significantincrease in mRNA level of both the genes (p<0.05) was detected. Thisincrease was particularly high for the scaffold with 2 mol % of Sr.

Enhanced proliferation of pre-osteoblasts over 14 days of investigationwas observed when Sr-substituted hydroxyapatite scaffolds were used(FIG. 7). In fact, an increase of strontium in the scaffold yielded amuch higher cell viability in the long term. These results imply thatnew bone formation could be successfully induced and sustained.

The scaffold also demonstrated the possibility of maintaining theosteoblastic phenotype during the two weeks of the investigation (FIG.8).

The behaviour of cells in contact with the strontium-substitutedscaffold was investigated also by observing osteoclast behaviour. Apreliminary morphological analysis was carried out to confirm andvalidate the model of osteoclastogenesis. Osteoclasts grown on thescaffold surface exhibit their typical morphology.

The relative gene expression of the principal marker involved inosteoclast activity and formation (Oscar, Integrin β3 and CatepsinK) wasevaluated (FIG. 9). The analysis showed a significant decrease in geneexpression over time of all the genes involved in the principalmolecular pathways of osteoclasts, thus indicating that the presence ofSr²⁺ ions in the scaffold inhibits osteoclast formation and activity.

In conclusion, the substitution of hydroxyapatites with Sr²⁺ionsproduced a biological effect on bone cells, specifically causing: i) asignificant inductive effect on MCSs osteogenic related genes; ii) aninductive effect on osteoblasts proliferation and iii) an inhibitoryeffect on osteoclasts activity.

In the case of implantation in a segmental bone defect, the new scaffoldis designed to present a central channel that extends in directionparallel to the main uni-directional porosity so to be exposed to thebone stumps as a guide for new bone marrow development (FIG. 1). Thechannel size is defined on the basis of the specific defect; however, tomaintain adequate strength the channel has a diameter in the range20-60% in respect to the whole scaffold width.

Example 4 Comparison of Biomorphic Hydroxyapatite of the Disclosure andHierarchically Structured Hydroxyapatite Known in the Art

A comparison test was made between a biomorphic hydroxyapatitemanufactured using the process of the disclosure and a hydroxyapatitescaffold obtained from the same native wood (rattan) according to theteaching of Anna Tampieri et al. in the Journal of Material Chemistry,2009, 19, 4973-4980. The rattan wood used in the process of thedisclosure has a length, measured along a direction in which a dimensionof the scaffold is maximum, equal to 2 cm. The rattan wood used in theprior art process has a length, measured along a direction in which adimension of the scaffold is maximum, equal to 1 cm.

After each step of the two processes, the specific surface area (SSA)and the pore distribution of the intermediate and final scaffolds wereanalyzed and compared (see FIGS. 10, 16, 19 and 20). In addition, afterthe carburization step the calcium carbide crystal dimensions of the twointermediate scaffolds were compared (see FIG. 13-bottom pictures).

The crystal lattice structure of the two calcium carbide scaffolds wascompared after the carburization step. The comparison is shown in FIG.14.

FIG. 10 shows a comparison of the pore distribution of the two calciumcarbide scaffolds obtained after the respective carburization steps,further compared with the pore distribution of the starting pyrolizedwood. The specific surface area of the two calcium carbide scaffolds arealso reported in the figure. The results show that only the calciumcarbide scaffold obtained after the carburization step of the disclosurepreserves the micro and nano-pore distribution of the rattan wood (poresdimension <1μm). Also the comparison of the two specific surface areasshow an improvement for the the scaffold according to the presentdisclosure.

FIGS. 11, 12 and 13-top two pictures show SEM immages of the two calciumcarbide scaffolds from which the better preservation of the native woodmicro and nano-porosity can be clearly seen.

FIG. 13-bottom two pictures show a comparison of the dimension of thecarcium carbide crystals. The calcium carbide obtained according to thepresent disclosure show granes with an average size of about 10 μm,while the granes obtained with the prior art process have an averagesize of about 100 μm.

FIG. 14 show a comparison of the crystal phase of the two calciumcarbide scaffolds, measured with x ray-XRD. The results show that thecalcium carbide obtained according to the present disclosure has both atetragonal and cubic crystal lattice, while the calcium carbide of theprior art has a tetragonal lattice only. The calcium carbide scaffold ofthe disclosure contains a higher amount of Ca(OH)₂ with respect to theprior art scaffold.

FIG. 15 shows SEM immages of the calcium oxide scaffold obtained afterthe respective oxidation steps. The pictures corresponding to thescaffold obtained according to the present disclosure preserves amicroporosity between the CaO granes, while in the prior art scaffoldthe microporosity is completely lost.

FIG. 16 depicts the pore distribution of the two calcium oxidescaffolds. The comparison clearly show that the micro and nano-porosityfraction obtained after the oxidation step is higher in the scaffoldaccording to the present disclosure as well as the specific surfacearea.

FIGS. 17 and 18 show SEM immages of the calcium carbonate obtained afterthe respective carbonation. The material according to the presentdisclosure show an extended fine structure, compared to the prior artwhere large crystals of calcite (up to about 50 μm) characterize thewhole structure. The large crystals cause the structure to break orcollapse during the phosphatization step.

FIG. 19 shows that the pore distribution of the two calcium carbonatescaffolds obtained after the carbonation step. The comparison of thepore distribution and the specific surface area show a result, similarto the one discussed above for the calcium oxide: the micro andnano-pore structure <1 um is maintained with the present process and anhigher SSA is obtained with respect to the prior art.

FIG. 20 shows the comparison between the pore distribution of the finalbiomorphic hydroxyapatite scaffold (after phosphatization) obtained withthe process of the disclosure and the final biomorphic hydroxyapatitescaffold of the prior art. A comparison of the respective SSA is alsoshown.

The results show that the biomorphic scaffold of the disclosure possessa higher micro and nano-porosity fraction than the prior art scaffold aswell as a higher specific surface area.

Example 5 Prior Art Process Applied to a Piece of Rattan Wood Having aLength, Measured Along a Direction in which a Dimension of the Scaffoldis Maximum, Equal to 2 cm

A test was made to demostrate that the prior art process (Anna Tampieriet al. in the Journal of Material Chemistry, 2009, 19, 4973-4980) doesnot allow manufacturing scaffolds having a length, measured along adirection in which a dimension of the scaffold is maximum, equal orgreater than 2 cm, i.e. scaffolds of clinical interest for boneregeneration.

To this purpose, a piece of rattan wood has been subjected to theprocess steps according to the conditions described in Tampieri et al.

FIGS. 21 show that even before the phosphatization step the scaffold canbreak down. FIG. 22 show that even if the scaffold survives the processsteps up to phosphatization, after phosphatization the scaffold breaksdown.

The test clearly shows that the scale up of a ceramic product is oftennot a straightforward operation; instead the process conditions need tobe changed (sometimes heavily changed) in order to prepare largerproducts, even when a process for making small ceramic product is knownin the art.

Example 6 Comparative Test

An in vitro study was performed with mouse mesenchymal stem cell(mMSCs). Gene expression profiling was analysed in order to test theover expression of specific genes involved in osteogenic differentiationinduced by a biomorphic hydroxyapatite scaffold obtained with theprocess of the present disclosure and a biomorphic hydroxyapatitescaffold obtained with the prior art process.

Sample Description

The two tested scaffolds are defined as follows:

Number Sterilization Sample Disk dimension of samples method Scaffold ofdoped Ca/P = 1.65 Ø: 8.00 mm, 5 EtOH + UV the present (disclosure) Mg/Ca= 1.64 L: 4.00 mm irradiation disclosure Sr/Ca = 0.59 Not Ca/P = 1.70 Ø:8.00 mm, 5 EtOH + UV doped L: 4.00 mm irradiation (disclosure) Scaffoldof Not- Ca/P = 1.77 Ø: 8.00 mm, 8 25 kGy γ-ray Tampieri et al. doped L:4.00 mm radiation (prior art) (GammaRad)

Results

The genes tested, related to both early (Runx2 and ALP) and late stage(OPN) commitment of osteogenic differentiation, seem to be upregulatedin the cells grown in all the disclosure scaffolds compared to the priorart scaffolds, with a significant difference for Runx2 and OPN(p≤0.0001). No differences were observed in BMP2 and Col15 geneexpression in all the samples tested, probably because BMP2, that is theupstream regulator of the differentiation pathway, after 14 days ofdynamic culture, it has already carried out its biological function assuggested by the upregulation of Runx2, Alp and OPN [1]. On thecontrary, Col15 is a very late marker related to the production ofmineralized bone matrix [2] and probably the time of culture was notsufficient to induce its expression. No differences were observedbetween the inductive effect of the doped or not-doped disclosurescaffolds (see FIG. 23).

FIG. 23 shows the relative quantification (2^(−ΔΔCt)) of gene expressionwith respect to the expression of the not-doped prior art scaffold usedas calibrator, after 14 days of mMSCs 3D cultured in dynamic conditionwith all the tested samples. Average and standard error of three sampleswere indicated. Statistical analysis was made by two-way ANOVA, followedby Bonferroni's post-hoc test and significant difference is indicated inthe graph: ****p≤0.0001.

From the above tests, it is possible to assert that the disclosurescaffolds show a higher inductive power on the expression of osteogenicrelated genes, with respect to the prior art scaffolds.

REFERENCES

[1] Arch Oral Biol. 2013 January; 58(1):42-9. doi:10.1016/j.archoralbio.2012.07.010. Epub 2012 Aug. 9. Leader genes inosteogenesis: a theoretical study. Orlando B, Giacomelli L, Ricci M,Barone A, Covani U.

[2] J Cell Physiol. 2012 August; 227(8):3151-61. doi: 10.1002/jcp.24001.Extracellular calcium chronically induced human osteoblasts effects:specific modulation of osteocalcin and collagen type XV. Gabusi E,Manferdini C, Grassi F, Piacentini A, Cattini L, Filardo G, LambertiniE, Piva R, Zini N, Facchini A, Lisignoli G.

1. A biomorphic hydroxyapatite scaffold obtained from a wood having atotal porosity of at least 20%, said porosity being measured aftersubjecting the wood to a step of pyrolysis, said scaffold having alength, measured along a direction in which a dimension of the scaffoldis maximum, greater than or equal to 2 cm.
 2. A biomorphichydroxyapatite scaffold according to claim 1 wherein the total porosityof the wood is between 60% and 95%.
 3. A biomorphic hydroxyapatitescaffold according to claim 1, having a compressive strength, measuredin the longitudinal direction, greater than 5 MPa.
 4. The biomorphichydroxyapatite scaffold according to claim 1, having a hierarchicallyorganized pore structure derived from the hierarchically organized porestructure of the wood from which it is obtained.
 5. The biomorphichydroxyapatite scaffold according to claim 1, wherein the wood isselected from the group consisting of rattan, pine, abachi, balsa, sipo,oak, rosewood, kempas and walnut wood.
 6. The biomorphic hydroxyapatitescaffold according to claim 1, wherein the hierarchically organized porestructure includes between 30 and 80%, of the total porosity, of poreshaving a diameter below 150 μm, the reminder to 100% of total porositybeing pores having diameter greater than 150 μm.
 7. The biomorphichydroxyapatite scaffold according to claim 6, wherein the wood israttan, and between 30% and 60% of the total porosity of the scaffold isdue to pores having a diameter ≤10 μm.
 8. The biomorphic hydroxyapatitescaffold according to claim 6, wherein the wood is rattan, and at least25% of the total porosity of the total porosity of the scaffold is dueto pores having a diameter ≤1 μm.
 9. The biomorphic hydroxyapatitescaffold according to claim 6, wherein the wood is rattan, and at least20% of the total porosity of the scaffold is due to pores that havediameter ≥150 μm.
 10. The biomorphic hydroxyapatite scaffold accordingto claim 6, having a specific surface area (SSA) >9 m²/g.
 11. Thebiomorphic hydroxyapatite scaffold according to claim 1, wherein saidhydroxyapatite is partially substituted with one or more ions selectedfrom the group consisting of magnesium, strontium, silicon, carbonate,sodium, potassium, silver, gallium and copper. 12-18. (canceled)
 19. Abone substitute comprising the biomorphic hydroxyapatite scaffoldaccording to claim
 1. 20. A process for producing a biomorphichydroxyapatite scaffold, comprising the steps of: 1) Pyrolysis: whereina native wood is heated at a temperature in the range of 600° C. to1000° C. under an inert atmosphere to yield a carbon template; 2)Carburization: wherein the carbon template is infiltrated with calciumin the vapour state at a temperature in the range 900 to 1200° C. and ata pressure of <1000 mbar, to yield a calcium carbide template; 3)Oxidation: wherein the calcium carbide template (CaC2) is heated in airat a temperature in the range of 750 to 1300° C., to yield a calciumoxide template; 4) Hydration: wherein the calcium oxide template isexposed to water, thus enabling water uptake in an amount of 1-25 mole%; 5) Carbonation: wherein the calcium oxide template is transformedinto calcium carbonate by heating at a temperature in the range of 500to 900° C., under a pressure in the range from 4 to 20 MPa; and 6)Phosphatization: wherein the calcium carbonate template is treated withat least one phosphate salt to yield the biomorphic hydroxyapatitescaffold.
 21. The process according to claim 20, wherein after step 1)the native wood has a total porosity of at least 20%.
 22. The processaccording to claim 20, wherein in step 1) the native wood is selectedfrom the group consisting of rattan, pine, abachi, balsa, sipo, oak,rosewood, kempas and walnut.
 23. The process according to claim 20,wherein prior to the pyrolysis step 1), the process comprises a step i)of selection and preparation of the native wood, wherein said nativewood is cut into a piece having a length, measured along a direction inwhich a dimension of the wood is maximum, that is greater than or equalto 2 cm.
 24. The process according to claim 23, wherein step i) ofselection and preparation of the native wood, comprises the steps of:providing a 3D model of a bone defect and, based on the 3D modelobtained, imparting to the native wood a shape corresponding to theshape of the bone defect.
 25. The process according to claim 20, whereinthe carburization step 2) is carried out with a Ca/C molar ratio, at thebeginning of the reaction, in the range of 1.10 to 2.50.
 26. The processaccording to claim 20, wherein the carburization step 2) is carried outat a temperature of 900 to 1200° C. and at a pressure <1000 mbar. 27.The process according to claim 20, wherein in the hydration step 4) thecalcium oxide template is exposed to water moisture, thus enabling wateruptake in an amount comprised in the range of 1-25 mole %.
 28. Theprocess according to claim 20, wherein the carbonation step 5) iscarried out according to one of the following thermal cycles: at aconstant CO₂ pressure of about 10 MPa, slowly increasing the temperatureat a value in the range of about 750-850° C.; at a constant temperatureof about 750-850° C. raising the pressure up to about 10 MPa; keepingthe pressure at about 4-6 MPa while raising the temperature up to about750-850° C. and subsequently increasing the pressure up to about 10 MPa.29. The process according to claim 20, wherein in the phosphatizationstep 6), the at least one phosphate salt is selected from the groupconsisting of ammonium phosphate, sodium phosphate, potassium phosphateand mixtures thereof.
 30. The process according to claim 20, wherein inthe phosphatization step 6) the calcium carbonate template is immersedin a water based solution comprising at least one phosphate salt, saidsolution having a phosphate concentration of 0.1 to 5M.
 31. The processaccording to claim 20, wherein the starting ratio of PO₄/CO₃ in thephosphatization step 6) is 1.5 to 5 times the theoretical stoichiometricvalue.
 32. The process according to claim 20, wherein thephosphatization step 6) is carried out in the presence of magnesium,strontium, silicon, carbonate, sodium, potassium, silver, gallium orcopper ions, or mixtures thereof.
 33. A biomorphic hydroxyapatitescaffold derived from wood obtainable from the process as described inclaim
 20. 34. A biomorphic hydroxyapatite obtained from a wood having atotal porosity of between 60% and 95%, said porosity being measuredafter subjecting the wood to a step of pyrolysis, wherein saidhydroxyapatite comprises a hierarchically organized pore structure and acompressive strength, measured along the longitudinal direction, greaterthan 5 MPa.
 35. A bone substitute comprising the biomorphichydroxyapatite scaffold according to claim
 33. 36. A method of treatingof a human or animal bone having a bone defect, comprising the steps of:providing a bone substitute comprising the biomorphic hydroxyapatiteaccording to claim 1 having a shape that corresponds to the shape of abone defect; implanting the bone substitute in the bone defect of thepatient.
 37. The method according to claim 36, further comprising thesteps of providing a 3D model of the bone defect and, based on the 3Dmodel obtained, imparting to the scaffold a shape corresponding to theshape of the bone defect.
 38. The method of claim 36, wherein said humanor animal bone is a bone subjected to mechanical loads.
 39. The methodof claim 36, wherein said human or animal bone is tibia, metatarsus,fibula, femur, humerus or radius.
 40. The method of claim 36, whereinsaid human or animal bone is a cranial bone-part, a spine bone or amaxilla-facial bone-part.